65,000-years of continuous grinding stone use at Madjedbebe, Northern Australia

Grinding stones and ground stone implements are important technological innovations in later human evolution, allowing the exploitation and use of new plant foods, novel tools (e.g., bone points and edge ground axes) and ground pigments. Excavations at the site of Madjedbebe recovered Australia’s (if not one of the world’s) largest and longest records of Pleistocene grinding stones, which span the past 65 thousand years (ka). Microscopic and chemical analyses show that the Madjedbebe grinding stone assemblage displays the earliest known evidence for seed grinding and intensive plant use, the earliest known production and use of edge-ground stone hatchets (aka axes), and the earliest intensive use of ground ochre pigments in Sahul (the Pleistocene landmass of Australia and New Guinea). The Madjedbebe grinding stone assemblage reveals economic, technological and symbolic innovations exemplary of the phenotypic plasticity of Homo sapiens dispersing out of Africa and into Sahul.

In 2017, high-resolution sampling of quartz-rich sediments for single grain OSL dating, combined with advances in OSL measurement technology and dating procedures, resulted in publication ) of 50 more accurate and precise age estimates than those previously published for Madjedbebe. OSL dating gives an estimate of the time since mineral grains were last exposed to sunlight. Full details of OSL and radiocarbon dating at Madjedbebe are provided in Clarkson et al. (2017) and Florin et al. (2021).
A ~2 m-thick archaeologically sterile sand unit (4.6-2.6 m below surface) underlies the first certain evidence for human occupation of the site, designated as Phase 1. The deepest samples published in Clarkson et al. 2017 were from ~2.9 m below surface giving a start date for this phase of 80.2 ± (7.2, 9.0) kyr ; the first and second error terms are the modelled age uncertainties at 95.4% probability, excluding and including the total systematic error, respectively. Sedimentation, however, commenced at a depth of ~4.6 m below surface. The revised age estimates for the samples collected for TL dating from the basal deposits by Roberts et al. (1990a) and Roberts and Jones (1994) suggest that the sand apron started forming 121 ± 18 kyr (1σ) ago, around the time of the last interglacial. The end date of this phase was calculated to be 71.0 ± (5.6, 7.3) kyr, which corresponds to a mean sediment accumulation rate of 4.0 ± 0.6 cm/kyr between 4.6 and 2.6 m depth. The uncertainty on this rate estimate is expressed at 68.2% probability and determined from the random errors only. This end date for Phase 1 also coincides with the transition from Marine Isotope Stage (MIS) 5 to 4, which may be significant as a palaeoclimatic control on the sand apron accumulation. Low densities of artefacts occur between about 3 and 2.6 m depth, but the first dense artefact band (Phase 2 in the Bayesian age model) occurs between 2.6 and 2.15 m below surface.
For this band, we obtained start and end ages of 65.0 ± (3.7, 5.7) kyr and 52.7 ± (2.4, 4.3) kyr, respectively, giving a mean sediment accumulation rate of 4.1 ± 0.8 cm/kyr over this depth interval. The latter was calculated from the modelled estimate of phase duration and the corresponding total random uncertainty at 68.2% probability; the same procedure was used for all subsequent phases. Phase 3 represents an archaeological unit, but with reduced artefact abundance.
The modelled start age for this unit is of 51.6 ± (2.4, 4.2) kyr, which is statistically consistent with the end age of Phase 2, suggests no significant time gap in sediment deposition. Phase 3 ended 28.1 ± (2.1, 2.8) kyr ago, indicating a much slower rate of sedimention over this period (2.6 ± 0.2 cm/kyr). The start of Phase 4 represents an increase in lithic abundance, beginning at 26.7 ± (2.2, 2.8) kyr ago and ended 13.2 ± (1.0, 1.3) kyr ago with a mean accumulation rate of 4.4 ± 0.4 cm/kyr during the lead up to and through the last glacial maximum. There appears to be a hiatus of 3.6 ± 0.9 kyr between Phases 4 and 5, coupled with a noticeable drop in artefact abundance associated with the latter, 25 cm-thick sedimentary unit (0.95-0.70 cm below surface). The modelled start and end ages for Phase 5 are 9.7 ± (0.8, 1.1) and 8.0 ± (1.0, 1.1) kyr, respectively, resulting in a mean sedimention rate of 15.7 ± 7.4 cm/kyr. Phase 6 represents a pulse of high lithic abundance in a 35 cm-thick band, which started 7.1 ± (1.0, 1.1) kyr ago. Phases 6 and 7 are represented by only four relatively imprecise OSL ages, so they are not well constrained by OSL dating. However, a number of 14 C ages from Phase 7 suggest that this unit accumulated within the last 600 years.
Further radiocarbon ages presented in Florin et al. (2021) confirmed that Phase 7 formed within the last 600 years and that a hiatus exists between Phases subsequently divided into Phase 7a, which formed 600 years ago, and Phase 7b, which formed in the last 150 years.
Many of the single-grain OSL equivalent dose (De) distributions include some grains with smaller and higher De values than those of the majority of grains, which we interpreted as evidence of small-scale mixing of the deposit. Furthermore, some of the OSL samples were collected in 5-cmdiameter tubes, which will also result in some time-averaging (approximately 1,250 years at an average sedimentation rate of about 4 cm kyr−1). For this reason, OSL age estimates were modelled as 'Phases' within the Bayesian framework, which assumes that the age estimates are unordered and uniformly distributed, so that any mixing within an archaeological phase (e.g., Phase 2) will not influence the model. This assumption was supported by the presence of stone artefact refits that showed horizontal and lateral movement within a phase but not between phases . The age of artefacts within a Phase can, therefore, be interpreted as falling somewhere between the modelled start and end ages of a Phase. For example, for Phase 2, the earliest and latest possible age estimates for human activity are 65.0 ± 3.7 kyr and 52.7 ± 2.4. It is, however, not possible to ascertain whether occupation occurred continuously throughout the entire unit, or whether it was clustered towards one end of the age range or close to the middle. Further confidence in the veracity of the age estimates were obtained from a series of interlaboratory comparisons, consistent age estimates obtained for both the 2017 and 1989 samples and from across several spatially distinct areas, and agreement with 14 C ages down to a depth of ~1.5 m presented in Clarkson et al. (2017) and Florin et al. (2021).
Contention around the age of Madjedbebe following publication has centred around whether artefacts could have moved down through sandy sediments over time as a result of bioturbation Introduction Ground-stone artefacts (i.e., stones that possess grinding or abrasive wear) include all stone items that have been either intentionally modified to a specific form through grinding (i.e., manufacture-ground); or used in the grinding, pounding or filing (abrading) of other materials (i.e., use-ground) (Adams 1994: 17;Odell 2004: 74-85). Manufacture-ground tools include both ornamental and utilitarian tools, such as polished stone, vessels and beads, as well as groundedge axes, adzes and bowls. Use-ground stones include all utilised grinding dishes, portable hand stones, stone "files" and large bedrock grinding patches. Functional analysis of use-ground stones (hereafter referred to as "grinding stones") involving the documentation of tool stone morphology, usewear and residues, can potentially indicate past grinding activities. Thus, the study of these implements from Madjedbebe in northern Australia provides a rich and unique source of archaeological evidence for the utilisation of plant, animal and other resources, since initial colonisation.
We distinguish two classes of grinding stone implements: (1) filing stones, which are used to process a material through direct contact (cf. "abraders" or "polishers" as described by Adams 1993by Adams : 64, 2002andHamon 2008: 1504); and (2) coupled stones, which are used in conjunction with another stone to process an intermediate material (Table S1).

Coupled stones
Coupled stones often include one large, stationary "lower" grinding stone and a smaller, active "upper" stone that is held in the hand(s) (Odell 2004: 78;Wright 1994: 239), which are used together to process an intermediate material. These artefacts may be previously unmodified stones, selected for size and shape, or they may have been manufactured, sometimes by grinding and flaking. The active stone is used in either a back-and-forth, rotary, or pounding motion to grind or otherwise process the material on the stationary basal stone. Hammerstones and other percussion/pounding tools such as mortars, anvils and pestles, may also be classified as coupled stones, as processing involves the contact of two stones during pounding and crushing activities (De la Torre 2013: 313;Odell 2004: 79;Wright 1994: 239). These tools often show traces of crushing, pounding, grinding and battering (Kraybill 1977: 493). Mortars are bowls or flatbottomed slabs with circular or oval depressions forming the concave receptacle that holds the material to be processed (Kraybill 1977: 491;McCarthy 1976: 63). Pestles are the accompanying pounding/grinding/crushing tools, which are usually fist-sized and rounded in section but can be elaborately carved. Pestles can simply be specially selected water-worn cobbles, and typically have crushing wear from impact with the mortar. Mortars and pestles are typically used to crack and pound hard materials such as seeds and nuts, which often contain a hard outer shell (e.g., Goren-Inbar et al. 2002;Peterson 1968). These implements may also be used to process other materials such as bone, shell and pigment (e.g., Liu et al. 2010;Peterson 1968;Van Peer et al. 2003).

Filing stones
Filing stones are used singly to process and shape a variety of materials such as stone, bone, wood, shell, ivory and ochre. Filing stones may exist as portable stone "files" (e.g., whetstones [hand-held implements used for grinding and sharpening stone axe blades, chisels and knives, McCarthy 1976: 60-61]; fish-hook files), as fixed features on rockshelter walls, and as large boulders and as outcropping bedrock, where they often occur in the form of circular or ovate grinding grooves (e.g., axe-grinding grooves). It is important to note that filing and coupled stones are not mutually exclusive; these tools may sometimes be used interchangeably to process multiple materials. For example, hammerstones and mullers can be used to polish wooden artefacts, and to process other materials on different lower stones (McCarthy 1976: 61). In our analysis, we refer to grinding stones as either a polishing, filing or coupled stone with reference to their design, dominant mode of use and most recent use (Table S1).

Distinguishing classes of grinding stones
Distinguishing between use-ground and manufacture-ground implements involves careful evaluation of the artefact design, manufacture traces, usewear and residues. Manufacture-ground implements such as ground-edge hatchets, axes and knives will typically display a bevelled edge with a finely abraded surface. Grinding wear on use-ground implements, on the other hand, will occur on the contact surface where grains have been altered or removed following contact with the processed material or upper stone. When only small fragments of a broken tool have survived, it may be very difficult to distinguish between use-ground and manufacture-ground traces.
Determining whether a use-ground stone should be classified as a coupled or filing stone depends on the artefact morphology and surface features recognised at various magnifications. For example, stones used for filing wood, shell or bone typically display a flat or concave cross section with no traces of stone-on-stone working-unless they were also used to sharpen or repair stone axes. In contrast, coupled stones such as mortars and pestles, and other lower and upper stones, will display stone-on-stone wear and will typically be concave and convex in section, respectively.
Broken fragments of lower stones have problematic morphologies with concave-convex portions. Although it is usually possible to distinguish between filing stones and coupled stones found archaeologically, the two tool classes are not necessarily mutually exclusive: it is possible to have a grinding stone which was used for multiple purposes, such as an upper or lower stone used as an impromptu filing stone to sharpen a stone axe. Broken pieces are, again, much more difficult to identify.  such as sandstone, they are usually porous with potential to preserve archaeological residues within the interstitial spaces of the stone matrix. For this reason, residue analysis on sandstone grinding stone implements, such as the majority of those recovered from Madjedbebe, may be particularly insightful for identifying past tool use.

Experimental reference libraries of usewear and residue traces
Functional interpretations for particular classes of stone tools can be validated using replicative (actualistic) and/or controlled experiments (Hayes et al. 2018a;Marreiros et al. 2020 Reference libraries of usewear on experimental grinding stones from Australian contexts are uncommon and scattered throughout the literature (e.g., Fullagar et al., 2012Fullagar et al., , 2015Fullagar et al., , 2017Hayes, 2015;Hayes et al., 2018bHayes et al., , 2020Spry et al., 2019), but have indicated that distinct patterns of usewear result from the working of different materials, modes and durations of use, and the stone material (Table S2). In order to create a usewear/residue reference library relevant to the study region, we conducted our own experiments using sandstone collected from near Madjedbebe and processed a range of materials. This research has been fully published by Hayes et al. (2018a) and forms part of the basis for our functional interpretations on the Madjedbebe grinding stones. In summary, we found that stone hardness greatly influences the formation of usewear and that harder sandstones (such as the stone from near Madjedbebe) typically sustain more developed use-polish during use than soft sandstones from other Australian locations, which wear more rapidly. We also found that microscopic usewear was also influenced by the class of grinding tool (upper, lower or filing stone), mode of use (pounding, grinding or filing) and the class of worked material (bone, stone, haematite, seeds, wood). Supplementary

Introduction
Residues are the remnants of material that have been transferred and attached to the artefact surface as a result of cultural and non-cultural processes. Residues from the surfaces of grinding stones are typically identified following extraction (usually with variable pipettes and distilled water or other solvents) and examination under high magnification using a transmitted light microscope. Biological staining agents may be applied to extracted materials to aid identification of unknown particles to confirm their origin (e.g., plant, animal and/or inorganic, see below).
Residue extractions were taken from the used and unused surfaces of each of the Madjedbebe grinding stones using distilled water and/or a tri-solvent mixture of acetonitrile, ethanol and distilled water and examined under an Olympus BX-51 metallographic microscope with objective lenses of x50, x100, x200 and x500 and polarizing filters. Identified particles are listed in Table   S4.

Staining of extracted residues
A common method for identifying residues in an extracted solution is with biological stains, a process that involves the application of various solutions to the extracted material and observing any subsequent related changes in appearance, typically under microscopic conditions. The applied solution, or "stain", will react with a certain component of the residue, turning the designated material a distinctive colour while leaving other constituent materials unaffected.
We selectively used seven different stains to confirm the identity of organic residues documented microscopically in water extractions (viewed under the transmitted light microscope) that had been sampled from the ground (and sometimes unground) surfaces of each grinding stone. Our selection of stains included those that work to indicate the presence of cellulose, lignin, damaged and undamaged starch, protein, collagen and keratin (e.g., hair and feathers), each of which are described alphabetically below. Stains were chosen on the basis of availability, cost and the procedure of application.

Congo Red
Congo Red (C32H22N6O6S2Not measured2) is a water-soluble dye that may be used as a general contrast stain for cellulose, amyloid fibrils, and damaged or gelatinised starches (Conn & Lillie 1969;Lamb & Loy 2005: 1439. The stain causes starch and cellulose to stain red while amyloid fibrils will stain green. The latter material is typically only stained in alkaline and acid buffer conditions (i.e., pH 2-4) where the stain is able to bind to carbohydrates, specifically amyloid (Chou et al. 2001: 218;Lamb & Loy 2005: 1439Ramesh & Tharanathan 1999: 347). At a neutral pH, cellulose fibres and damaged or gelatinised starch may be stained in isolation. Both cellulose and starch are composed of the same monosaccharide molecule (glucose), however, differences between the bonds linking the glucose units of both cellulose and starch account for their separate structures and properties (Lamb & Loy 2005: 1434. Undamaged or unaltered starch grains will not become stained with Congo Red as they are hydrophobic and, therefore, will not take up the stain. Alternatively, any alteration of the compact and regular arrangement of the starch layers following heating (e.g., cooking) or mechanical damage (e.g., grinding and pounding) will cause the stain to penetrate into the grain and stain the amylose content within the damaged/altered grains, turning them red. The Congo Red solution was tested prior to application on archaeological artefacts by applying a small amount of stain (up to 5 µL) on heated corn starch in which a positive colour change (red) was identified.

Iodine Potassium Iodide
Iodine Potassium Iodide (IKI) was used to stain intact undamaged starch granules because it is known to bind to the amylose polymers (made up of glucose units) within the starch (Banks & Greenwood 1975: 67;Yeung 1998: 132). The stain provides an immediate colour change causing undamaged starch grains to turn yellow (Plate 5.1b). In time, the starch will turn a permanent dark-blue/black colour (Banks & Greenwood 1975: 67;Evert 2006: 53). The IKI solution was tested prior to application on archaeological artefacts by placing ~5 µL of stain onto a prepared slide containing potato starch in which a positive colour change was identified. The IKI stain also displayed a positive colour change with cellulose material mounted on other experimental slides (instantly staining purple).

Methylene Blue
Methylene Blue (C16H18N3SCI) is a water-soluble dye that may be used to highlight non-lignified cell walls such as cellulose fibres within plant material (Cutler et al. 2008: 180;Wilson 1907: 647). The stain binds to the acidic pectins on the cellulose cell wall that are stained various shades of blue (Plate 5.1c) (Lillie 1976: 425;Stadelmann & Kinzel 1972). The colour and intensity of the highlighted cellulose fibres is related to the purity of the material: the darker the blue, the purer the cellulose. Methylene Blue solution was tested prior to application on archaeological artefacts by placing ~5 µL of stain onto a prepared slide containing tissue paper in which a positive colour change was identified. A colour change was not identified on prepared collagen slides.

Orange G
Orange G (C16H10N2Not measured2O7S2) is an acidophilic dye used to stain protein and highlight various animal fibres including collagen and keratin, which typically stain orange (Plate 5.1d).
The associated change in colour occurs as the stain binds with proteins within the target materials. When used in conjunction with other stains, such as acid fuchsin and malachite green, pollen granules may be stained red (Alexander 1969;Lillie 1976: 121). The Orange G solution was tested prior to application on archaeological artefacts by placing up to 5 µL of stain onto a prepared slide containing a thin section of turkey meat in which a positive colour change was identified. No colour change was observed on slides containing plant materials.

Phloroglucinol
Phloroglucinol (C6H6O3) is a water-soluble dye used as a general contrast stain for lignin. The stain reacts with structures within the xylem and sclerenchyma of plant cells to turn the substance red (Plate 5.1e) (Cutler et al. 2008: 180;Jensen 1962). However, experimental staining of plant cells using Phloroglucinol of an altered pH, caused lignified tissues to turn a yellow-brown colour. Subsequent analyses of archaeological residues were confirmed for lignin if the latter colour change was observed.

Rhodamine B
Rhodamine B (C28H31CIN2O3) is a basic dye used to highlight the presence of animal fibres such as hair, feathers and collagen, binding with proteins to allow the target material to turn a pink/purple (Plate 5.1f) (Liisberg 1968;Wessley et al. 1981). In general, Rhodamine dyes are water-soluble and are most commonly used in applications of fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy and ELISA. The Rhodamine B solution was tested prior to application on archaeological artefacts by placing ~5 µL of stain onto a prepared slide containing highly degraded hair removed from an ancient, Native American leather artefact in which a positive colour change was identified.

Safranin
Safranin (C20H19CIN4) is a staining solution used as a contrast stain to highlight chromosomes, nuclei, lignin, and cell walls. A positive colour change will occur in the presence of the latter two features as the stain reacts with the xylem and sclerenchyma, causing lignified cell walls to turn pink while lignified fibres turn red (Plate 5.1g) (Srebotnik & Messner 1994). The Safranin staining solution was tested prior to application on archaeological residues by placing up to 5 µL of stain onto a prepared slide containing plant cells from a plant stem in which a positive colour change was identified. A colour change was not identified on prepared collagen slides. Because the Safranin stain will highlight other materials such as pollen grains, mitochondria and various animal cells, Phlorogluconol was the preferred stain used for the identification of lignin. The Safranin stain was used only when Phlorogluconol was not available.  (Evert, 2006). Reichert (1913) was first to explore whether plant taxa could be identified on the basis of starch grains and noted that the size and shape of different starches can often be (broadly) correlated with the source plant taxa. Over the past two decades there has been an increasing number of reports attributing starch grains to specific plant taxa based on features such as grain shape, maximum length through hilum, and the presence or absence of some morphological features such as fissures, surface 'folds', or lamellae and/or vacuoles (e.g., Liu et al., 2010;Louderback et al., 2017;Musaubach et al., 2013;Piperno et al., 2000). One of the most common effective measures for discriminating between starch grains in comparative reference collections descriptions ishas been the measure of maximum length through the hilum, with a population sample of at least 100 for >100 grains, and the minimum and maximum measurements presented as the size range for that species. More recently, Field (2015, 2018) and Field et al., (2016), developed a technique methodology based on geometric morphometric analyses, to record and evaluate specific starch grain features attributes for to enable a more reliable and accurate taxonomic identification. In this method, eachEach starch grain is photographed and is manually traced via a digitizing tablet, and with the hilum position, presence of absence of lamellae, fissures, vacuoles and grain facetis was noted located via a digitizing tablet using micrographs of individual grains. A range of geometric attributes can then be extracted from the tracedunkown starch grains shape that are then be compared statistically across large populations of grains derived from the reference species to allow for more reliable taxonomic identifications (see Hayes et al., 2021).

Extraction and analysis of recovered starch grains
Following pipette extractions from the ground surfaces of grinding stones, residue sampling for ancient starch analsyis was undertaken on a selection of 27 grinding stones (Table S5). The grinding stones were either partially or completely immersed in a beaker of distilled water that was placed in an ultrasonic bath for 2 min. The samples were then transferred to individual 50 ml Falcon tubes and centrifuged for 3 min at 3000 RPM. Starch (and phytoliths) were isolated from the residue sample using heavy liquid separation (Sodium polytungstate, Specific Gravity 2.35) by centrifugation for 15 min at 1000 RPM. Following rinsing in water and centrifugation, samples were slide-mounted in distilled water and complete slide scans were undertaken using a Zeiss Axioskop II brightfield transmitted light microscope under Differential Interference Contrast (DIC). Images were captured using a Zeiss HRc camera, and archived using Zeiss Axiovision software. Starch grain images were subsequently traced with a WACOM Intuos Pen Tablet (CTH-480) using a graphical user interface (GUI) developed in MATLAB by Adelle Coster (MATLAB Release 2014b, The MathWorks, Inc., Natick, MA, USA) and the size of the grains determined. Raw counts of starch and associated size ranges are shown in Table S5.

Extraction and analysis of recovered starch grains
A reference collection of well-curated starches of known taxonomic origin and documented as being economically important, was compiled from for the relevant geographic region where Madjedbebe is found was constructed. There are many starch yielding plant species from northern Australia that were exploited by Aboriginal people. Some were eaten raw, others lightly roasted while others were processed by pounding or grinding before cooking (e.g., Thomson, 1939;Wightman and Smith, 1991;Russell Smith et al., 1997, 1985Fox and Garde, 2018). Some starchy species not generally reported as pounded or ground were also considered as it is well documented that plant foods were prepared for the very young and the infirm. A range of ethnographic sources were consulted to compile a list of relevant plant species (e.g., Smith, 1991;Russell Smith et al., 1997;Meehan et al., 1978;Chaloupka and Giuliani, 1984;Fox and Garde, 2018;Jones and Meehan, 1989;Wightman and Smith, 1991 Identification of unknown starch grains to plant taxa is not presented here and will be published in detail shortly.  (5) the Hemastix® test, for the detection of ferrous iron (haem and haemoglobin). These tests were selected for application as they allow for the detection of a wide range of organic materials (in addition to inorganic iron-rich mineral crystals), including various plant and animal tissues.
Importantly, the selected tests were also able to be modified so that they may be performed as micro-biochemical tests (requiring less residue material) and analysed using a spectrophotometer. However, the selected tests are only specific for a group of compounds (e.g., protein, carbohydrates), and are unable to identify individual compounds (e.g., collagen, myoglobin). These tests are therefore less sensitive than other methods of residue characterisation such as GC-MS, and have only been used here as an initial screening test for the presence of these specific groups of organic compounds.
Five of the biochemical tests (all but the Hemastix test®) required the application of a colour reagent, which is added to a small portion of the extracted residue solution (see below). The intensity of a subsequent colour change can indicate the concentration of a given compound within the solution. Importantly, all six biochemical tests were able to be modified so that they could be performed as micro-biochemical tests (requiring less solution from the extracted residue mixtures) with subsequent colour changes detected using a spectrophotometer, an instrument that can measure the intensity of light (the number of photons) absorbed after it passes through a very small volume of solution (<5 μL).
The biochemical test reagents (described individually below) were added to individual portions (<5 μL) of solution extracted from the ground surfaces of each grinding stone using either distilled water or a solvent mixture of acetonitrile, ethanol and water in equal parts. To assess the possibility of environmental or other contamination, all accompanying sediment samples (made into solution by mixing with distilled water) and many extractions from the unground surfaces were also tested. Following the application of the biochemical reagents to the residue mixtures, the new solution was observed for a subsequent reaction using an Epoch TM Multi-Volume Spectrophotometer System (described in the main text). Positive readings were determined from a set of standard measurements made on low concentrations of blood protein, corn starch, cooking oil and a combination of sucrose and glucose. The readings from these measured standards (Table S6A) were considered the minimum value for the detection of proteins, starch, fatty acids and carbohydrates, respectively. The specific methods of each test are described below; the measured values for each residue sample are listed in Table S6B.  et al. (1989) and Kruger (1994). Five micro-litres of water-extracted material was added to 25 μL of Bradford Assay reagent (100 mg of Coomassie Blue G250, 50 mL of 95% ethanol and 100 mL of 85% phosphoric acid; made to 1 L with distilled water) and mixed for 20 min at 1,000 RPM at 25°C. Absorbance was then read for 2 μL of this solution at 595 nm. Diphenylamine (MW 169.22), 5 mL Glacial Acetic Acid and 0.125 mL sulphuric acid) and heated for 10 min at 80°C. Following heating, 2 μL of solution was measured for absorbance at 595 nm.

Phenol-Sulphuric Acid test
The Phenol-Sulphuric Acid (PSA) test is credited as the easiest and most reliable method of carbohydrate detection (Masuko et al. 2005: 69). The test is often used to measure the neutral sugars present within oligosaccharides, proteoglycans, glycoproteins and glycolipids, and was selected as an additional method of carbohydrate detection in the analysis of the MJB and Lake Mungo grinding stones. Five micro-litres of the water-extracted residue solution was mixed with a PSA solution (5 μL 4% Phenol and 25 μL Sulphuric acid). The mixture was left for 10 min at room temperature to ensure adequate binding of the PSA solution to any potential carbohydrates.
Following resting, 2 μL of solution were read for absorbance at 490 nm.

Iodine potassium iodine (IKI) test
The presence of starch (intact and gelatinised) was assessed using the IKI biochemical test (McCready & Hassid 1943). This test was selected owing to the high probability that some of these artefacts were used in the processing of plant materials, and such a test will indicate the presence of starch even if they are unable to be visually identified. Five micro-litres of the waterextracted material removed from each of the used surfaces were mixed to a solution of 5 μL potassium iodide (KI) (0.12M) and 5 μL of iodine (I) (0.01 M). Samples with <5 μL of extraction available were added to smaller portions of KI and I, ensuring that the ratio remained at 1:1:1.
Two micro-litres of solution were read for absorbance at 595 nm.

Falholt test
Fatty acid compounds were detected following the application of the Falholt test (Falholt 1973).
This test was considered highly useful for the analyses of grinding stones that may have been used to process materials with a high fatty acid content, such as seeds. Because fatty acids are also present in animal tissues and oily excretions of the hands, further characterisation of specific fatty acid compounds is required to determine the residue source. Residue extractions were freeze-dried for 48 hours so that any additional liquid was removed, and then resuspended in would not be made after 1 min as the pad can auto-oxidise and change colour, creating a falsepositive result. Colour change was ranked on a scale of 0-5 as recommended on the Hemastix package: 0 representing no change in colour; 1 for a speckled colour change and 2-5 for a broad colour change ranked on increasing darkness. These correspond to negative, slight trace, trace, small, moderate, and large traces of haemoglobin, respectively. Any sample that displayed a positive reaction (i.e., colour change ranking from 1-5) was then assessed for contamination by testing the corresponding sediment sample -sediments were submerged in distilled water and the suspended solution was assessed for potential residues causing a positive Hemastix® reaction. Because several other materials found within the burial environment are known to react with Hemastix, for example plant material and metal ions present within the soil (see which increases the specificity of the test and eliminates the reaction of metal ions within the tested sample (Matheson & Veall 2014). This mixture aims to eliminate any environmental or metal (including haematite) residues that cause a positive reaction in the Hemastix®. A sample that tests positive following the addition of the EDTA solution is likely to contain haemoglobin.
In this way, we can determine which artefacts are likely to contain blood and haematite residues.

Absorbance spectroscopy
Absorbance spectroscopy is a method that can be used in residue analysis to measure and record the spectra of absorption within a solution, allowing the chemical groups of the major constituents within a mixture to be recognised. The method involves the collection of spectral data from a sample that is exposed to a wide spectral range (e.g., 200-900 nm) to produce a "fingerprint" spectrum. The patterning of the spectra permits identification of the various residue components. One advantage of this technique is that analysis requires only a small portion of the residue solution (approximately 2 ɥL) and the majority of residue components can be quantified. However, because this method is less specific (identifying groups of compounds only), and less sensitive than other methods of residue characterisation such as GC-MS, it is more suitable as an initial screening test to confirm the presence of organic materials. In our analysis of the grinding stones from Madjedbebe, we generated absorbance spectra for residue samples extracted from both the ground and unground surfaces using mostly distilled water (Table S6B).

Spectral peaks
Nine peaks within the measured spectra range of 200 and 900 nm were of particular interest; these included: (1)  indicating the presence of plant components such as chloroforms and keratins (Hayes, 2015). The identification of distinctive "shoulders" or peaks in the resulting spectra indicated a positive reading.

Introduction
Gas chromatography-mass spectrometry (GC-MS) is a method of residue characterisation that allows non-visible trace elements in residue mixtures to be identified through separation (chromatographic) and identification (mass spectrometric) techniques. The procedure involves separation of molecules within a sample that then are ionised, detected and measured separately to provide information of the biomolecular components within the residue mixture. The presence of certain biomolecules or combinations of biomolecules (the 'chemical fingerprint') within the sample can be related to the compositions known for other organic materials to determine the residue origin may be determined (Evershed, 2008). Organic compounds such as lipids, terpenes, terpenoids, alkanes, proteins and carbohydrates are often the analytes of interest in determining the origin of unknown residues (e.g., Barnard et al., 2007;Buonasera, 2007;Evershed 2008;Dunne et al., 2012;Villa et al., 2015;Luong et al., 2017Luong et al., , 2018Luong et al., , 2019.

Biomolecular components of extracted residues from the Madjedbebe grinding stones
Analysis of residue mixtures sampled from the ground surfaces of the grinding stones and fragments from Madjedbebe revealed a range of biomolecular components, including plant and animal-specific compounds (Table S7). Residue mixtures that were abundant in fatty acids and glycerides indicate that the contact material had a high oil content. Monoglycerides are naturally found in seed oils and have been identified on a number of artefacts and often indicate the processing of seeds, nuts and oily fruits (e.g., olives). As there are no oil-producing fruit in the region, the presence of these residues on grinding stones and fragments from northern Australia support the processing of seeds and/or nuts that grow locally in the area. Fats that are derived from animals contain a slightly greater variety of fatty acids and glycerides and thus we were able to distinguish the presence of animal residues from those that may originate from seeds and other oily plants/plant parts (e.g., nuts, leaves and oil-producing fruits). Conversely, carbohydrate-based storage organs such as tubers, rhizomes, corms and many fruits (excepting oil-producing fruits such as olives) contain very few fatty acids and virtually no glycerides.
Several samples also contained ascorbic acid (vitamin C) found in tubers, fruit, kernels, seeds and nuts. The presence of these residues on grinding stones and fragments from northern Australia support the processing of seeds and/or nuts which grow locally in the area. Table S7: List of compounds detected with GC-MS in residue extractions sampled from the Madjedbebe grinding stones and fragments. Compounds with known origins (library matches) are listed in bold text.